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Laboratory of Structure Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026, P.R. of China
Y. Z. Xiao
J. Wu
Y. Z. Hong
Y. P. Wang
Laboratory of Microorganism and Gene Technology, School of Life Sciences, Anhui University, Hefei, Anhui, 230039, P.R. of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71 454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5–7.5. The Km values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 µM. The corresponding Vmax are 504.0, 1910.0, 117.4 and 159.0 µM min-1 mg-1. In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.
Key words: Aromatic compounds, 3,5-dihydroxytoluene, guaiacol, laccase isozyme, o-toluidine, Trametes sp. AH28-2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Bourbonnais et al 1995), morphogenesis (Zhao et al 1999), pigment synthesis, sporulation (Leatham et al 1981), phytopathogenesis and fungal virulence (Rigling et al 1993, Williamson 1997). Laccase is considered to be a potentially important industrial enzyme. Laccase can be applied extensively in many fields, which include waste detoxification and textile dye transformation (Rama et al 1998, Abadulla et al 2000, Fukuda et al 2001), delignification of lignocellulosics and cross-linking of polysaccharides (Poppius-Levlin et al 2001), upgrading of wine quality (Lante et al 1992, Servili et al 2000), removal of fermentation inhibitors to increase yield of ethanol (Larsson et al 2001), improvement of drug analysis (Bauer et al 1999), as well as construction of new-type energy producing devices and new enzyme sensors (Freire et al 2001).
Laccase typically contains 15–30% carbohydrate. Laccase usually has an acidic isoelectric point and has a molecule mass of 60–90 kDa. Laccase is encoded by a family of genes and produced in the form of multiple isozymes. It has been proven that genes encoding laccase isozymes were differentially regulated (Soden et al 2001). Some are constitutively expressed, others are expressed upon induction by aromatic compounds relevant to lignin, such as kraft lignin, xylidine, veratryl alcohol, etc. (Gianfreda et al 1999, Dekker et al 2001). The yields of laccase from Pycnoporus sanguineus can be increased 50 times using 20 µM xylidine (Pointing et al 2000). Syringaldazine can induce the constitutive form of laccase from Coriolus hirsutus to a 10-fold (Koroljova-Skorobogat'ko et al 1998). Other major factors that can affect laccase synthesis include concentrations and types of carbon and nitrogen sources and metal ions such as copper in the media (Galhaup et al 2001, Levin et al 2002, Baldrian et al 2002).
Trametes sp. AH28-2 is a white-rot basidiomycete that selectively degrades lignin when grown on wood and reduces the level of chemical oxygen demand (COD) in wastewater produced in the process of straw pulping (Wu et al 2002). Preliminary studies indicate that this microorganism can be induced by kraft lignin to secrete high levels of extracellular laccase (Xiao et al 2001, Zhang et al 2002). The extracellular laccase contains at least two kinds of laccase isozymes, namely laccase A and laccase B. Laccase A, which accounts for about 85% of the total activity, has been purified and characterized (Xiao et al 2003). However, laccase B has not been successfully purified due to its low expression under normal culture conditions. Here we have tried to induce laccase production with different aromatic compounds and by optimizing the culture conditions. We have shown that several potential laccase inducers can induce Trametes sp. AH28-2 to synthesize laccase differentially; 3,5-dihydroxytoluene selectively can enhance laccase B production. Here we report the successful purification and characterization of laccase B. Our study shows that it is a glycolyated laccase that contains copper and is different from laccase A.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Microorganism –
Trametes sp. AH28-2, which was deposited in the culture collection of the School of Life Sciences, Anhui University, China, was found to produce extracellular laccase on an optimized medium (Xiao et al 2001). Aspergillus oryzae (CGMCC No. 3.2825) and Gloeophyllum trabeum (CGMCC No. 5.98) were obtained from China General Microbiological Culture Collection Center (CGMCC). Stock culture was maintained on CPDA medium agar slant at 4 C and inoculated once every 3 months.
Growth conditions and induction of laccase – Five or six cylinders (10 mm diam) of Trametes sp. AH28-2 grown on CPDA plates were inoculated into 100 mL liquid medium in 250 mL Erlenmeyer flasks. Every liter of liquid medium contained 10.0 g cellobiose, 5.0 g glucose, 1.5 g L-aspargine, 1.0 g tryptone, 1.0 g KH2PO4, 0.5 g MgSO4·7H2O, 0.01 g CaCl2, 0.001 g FeSO4·7H2O, 0.1 g Na2HPO4·5H2O, 0.002 g CuSO4·5H2O, 0.0275 g Adenine and 50 µg Vitamin B1. The pH was adjusted to 5.6 with phosphoric acid before sterilization. The culture was incubated at 28 C and shaken at 110–120 rpm. After 72 h, the culture was homogenized using a sterilized blender. Ten mL culture was inoculated to 500 mL fresh medium. After another 72 h, inducer was added to stimulate laccase production. The culture was collected every 24 h 24–144 h after induction.
Cocultivation – A. oryzae or G. trabeum was cultured 48 h and homogenized well, then inoculated into a 60–72 h grown culture of Trametes sp. AH28-2. The coculture supernatants were collected at the same time as that of Trametes sp. AH28-2 alone.
Laccase activity assay –
The laccase activity was measured in a 5.0 mL reaction volume containing the indicated concentration of laccase, 1 mM guaiacol and 50 mM succinic acid-NaOH buffer (pH 4.5). Oxidation of guaiacol was determined by the increase in A465 (
= 12 000 M-1 cm-1) (Xiao et al 2001). One unit was defined as the amount of the laccase that oxidized 1 µM of substrate per min. Assays were carried out independently in triplicate.
Enzyme purification – Unless otherwise stated, all procedures were performed at 4 C. A 1200 mL culture was filtered through six layers of gauze, centrifuged at 6000 g for 30 min and concentrated to 60 mL in MinitanTM Ultrafiltration System with a low binding regenerated cellulose membrane (Millipore, Bedford, Massachusetts). The concentrate was centrifuged at 12 000 g for 20 min then dialyzed against buffer A (10 mM citrate-Na2HPO4, pH 6.0) overnight, followed by centrifugation again. The supernatant was applied to a DEAE-Sepharose FF column (10 x 200 mm, Amersham Pharmacia Biotech, Sweden) that was pre-equilibrated with buffer A. The column was rinsed with 80 mL buffer A to remove melanin and polysaccharide, then eluted with linear gradient 0–0.3 M (NH4)2SO4 in buffer A, with a flow rate of 0.8 mL min-1. The fractions containing laccase activity were pooled and concentrated to 1.5 mL with an Amicon ultrafiltration stirred pressure cell equipped with 47 mm 30 000 molecular-weight cut-off filter. The concentrate was subjected to gel filtration in a Hiload 26/60 Superdex 200 prep grade column (Amersham Pharmacia Biotech, Sweden) that was pre-equilibrated with buffer B (10 mM citrate-Na2HPO4, 0.15 M (NH4)2SO4, pH 6.0). The active eluants were pooled and dialyzed against buffer A.
Native-PAGE and SDS-PAGE – Enzyme purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis was performed on 10% polyacrylamide gels. Proteins were visualized by staining with Coomassie brilliant blue R250. After Native-PAGE, the gel was incubated at 25 C in citrate-Na2HPO4 buffer (pH 6.0) containing 2.0 mM guaiacol to identify laccase components. The intensity of each isozyme's (laccase A and laccase B) staining bands, which were separated by PAGE gel, was monitored by using Eagle Eye II Still Video System (Stratagene, U.S.A.) supported by Eaglesight software to calculate the proportion of laccase A to laccase B.
Molecular mass –
The molecular mass of denatured laccase was estimated on a SDS-PAGE gel. The apparent molecular mass of active laccase was determined by FPLC with gel filtration on a superdex 200 HR10/30 prep grade column (Amersham Pharmacia Biotech, Uppsala, Sweden). MALDI-TOF-MS spectra were determined by using a model BIFLEXTM III MALDI-time-of-flight mass spectrometer (Bruker Co.) with 
-cyano-4-hydroxycinnamic acid as matrix.
Isoelectric point determination – The isoelectric point was estimated by isoelectric focusing (IEF) using a Bio-Rad Fast Gel System with Bio-Rad wide-range ampholytes (pH 3.0–9.0) and Pharmacia low-range ampholytes (pH 2.5–5.0).
Kinetic measurements –
All the laccase catalytic assays were done at 25 C. Initial velocity was measured in 3-mL glass cuvettes with 1 cm path lengths. Reactions were initiated by adding laccase. Initial rates were calculated from the linear portion of the progress curve. The wavelength used for determining catalytic reaction velocities was 420 nm for ABTS ( = 36 000 M-1 cm-1), 465 nm for guaiacol ( = 12 000M-1·cm-1), 530 nm for syringaldazine ( = 65 000 M-1 cm-1) and 468 nm for DMOP ( = 49 600 M-1 cm-1).
Protein electroblotting and sequencing – Purified laccase was electroblotted directly from the SDS-PAGE gel onto a polyvinylidene difluoride membrane (Sequi-Blot PVDF, Bio-Rad) and located by Coomassie blue R-250 staining. The band on the PVDF membrane corresponding to the laccase B protein was excised and subjected to Edman degradation. The N-terminal amino acid sequence was determined using an Applied Biosystems Procise 491 automatic sequencer (Applied Biosystems, Foster City, California).
Other methods – The laccase UV-absorbance spectrum was scanned from 200 to 800 nm at room temperature on model UV110 Spectrophotometer (Peking Ruili Co., China). The copper content of laccase was determined using atomic absorption spectroscopy with a PE 3100 apparatus (Perkin Elmer, U.S.A.) and Inductively Coupled Plasma Mass Spectroscopy (ICPMS) (PlasmaQuad 3, VG Elemental, Thermo Jarrell Ash Corp., U.S.A.). Protein concentration was determined using BCA assay kit (HyClone-PIERCE).
N-glycosidase F (Roche Diagnostics GmbH, Germany) was used to determine the carbohydrate content of laccase by comparing the migration of treated enzyme with that of untreated enzyme on a SDS-PAGE gel. Thermal stability was assessed between 4 and 60 C in citrate-Na2HPO4 buffer. The optimal pH for laccase was determined in citrate-Na2HPO4 buffer with guaiacol as substrate. Inhibition studies were performed using guaiacol as substrate in citrate-Na2HPO4 buffer (pH 6.0). To assess stability of laccase B at different pH values, laccase B was incubated 24 h at 25 C in citrate-Na2HPO4 buffer (pH 2.5–8.0) or Na2CO3-NaHCO3 buffer (pH 9.2–10.7). Then, the activities were assayed as mentioned earlier.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and 74 kDa on SDS-PAGE respectively (Fig. 1C). The ratio of laccase A to laccase B varied with induction by different compounds. When induced with o-toluidine, 2,5-xylidine, vanillin, syringic acid or 2,4-diaminotoluene, the dominant laccase component identified was laccase A. The activities of laccase A and laccase B were induced almost equally by ferulic acid or guaiacol. It is interesting to note that the dominant component induced by 3,5-dihydroxytoluene was laccase B, which accounted for more than 80% of the total activity in the supernatant (Fig. 2).
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Laccase B, a glycosylated copper laccase –
Laccase B production was induced selectively by 3,5-dihydroxytoluene. To purify laccase B, the supernatant induced by 3,5-dihydroxytoluene was concentrated and dialyzed, then applied to a DEAE-Sepharose FF column pre-equilibrated with buffer A. After that, the column was eluted with linear gradient of 0–0.3 M (NH4)2SO4 in buffer A. The activity of laccase B was detected at 0.1 M (NH4)2SO4, then the laccase B fraction was concentrated and dialyzed before gel filtration in a Hiload 26/60 Superdex 200 prep grade column through linear elution. Finally, the activity of laccase B was collected at the elution volume of about 150 mL. By SDS-PAGE examination, this component was purified to homogeneity (Fig. 1C). The purification fold was estimated to 40.2 with a yield of 19.3% (Table III). Purified laccase B in solution appeared blue. The UV spectrum of laccase B showed a broad peak at about 600 nm and a shoulder peak at about 330 nm. The 280/600 nm absorbance ratio was determined to be 21. Each molecule of purified laccase B was measured to have approximately 4.1 copper atoms by AAS, and 4.0 by ICPMS. Therefore laccase B was a typical copper-containing blue protein with four copper atoms: one type I, one type II and two type III (Thurston 1994). The molecular mass of laccase B was estimated to be 72 kDa by gel filtration chromatography and determined to be 71 454 Da by MALDI mass spectrum. After deglycosylation treatment with N-glycosidase F, the molecular mass of laccase B was shifted from 74 kDa to 55 kDa (Fig. 1A) showing that laccase B was a monomeric glycoprotein with 25% carbohydrate content.
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Laccase B could catalyze the oxidation of aromatic compounds under standard assay conditions (Table IV). Syringaldazine, ABTS, guaiacol and DMOP were preferred as substrates of laccase B. The relationship between enzyme activity and substrate concentration was of the Michaelis-Menten type. The apparent Km value of the enzyme for syringaldazine was estimated to be 28.0 µM, which was lower than those of the other compounds tested. The apparent Km values, determined with ABTS, guaiacol and DMOP as substrates, were respectively 177.0, 1249.0 and 109.8 µM. Corresponding Vmax values were 504.0, 117.4 and 159.0 µM min-1 mg-1. The kcat values for the above substrates ranged from 140.0 to 2277.7 s-1. This suggested that ABTS might be an effective substrate of this enzyme.
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N-Terminal amino acid sequence of laccase B – The unique sequence of the protein sample was determined by the Edman method. The sequence of first 15 residues at the amino terminus was AIGPVTDLTISNADV, which is identical to that at the amino terminus of the lcc5 mature protein from T. villosa (Yaver et al 1996).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), has been found to be incapable of stimulating Trametes sp. AH28-2 in producing laccase. Among all the compounds we tested, o-toluidine, rather than 2,5-xylidine, was the most efficient and 2,5-xylidine was the most suitable inducer in most cases (Pointing et al 2000). One of the interesting discoveries is that inducers with different structures can induce Trametes sp. AH28-2 to synthesize laccase isozymes of different composition. Guaiacol can induce Trametes sp. AH28-2 to synthesize laccase A and laccase B almost in equal proportion. O-toluidine can induce laccase A much more than laccase B. However, 3,5-dihydroxytoluene mainly can induce laccase B. As for their chemical structures (Fig. 5), although all three compounds are aromatic with one benzene ring in common, they have various substituted groups, respectively. Guaiacol has a phenolic group and a methoxy group at ortho position, o-toluidine has an amino group and an ortho methyl group, while one methyl group and two phenolic groups are substituted in meta positions of the benzene ring in a 3,5-dihydroxytoluene molecule. How the three compounds with different chemical structures affect the composition of laccase isozymes in Trametes sp. AH28-2 is not yet clear.
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). Therefore, laccase may function as a defense mechanism against oxidative stress. Laccase reactions, by consuming oxygen, are expected to disfavor redox cycling of quinines with oxygen, whereas the autoxidation of hydroquinones and semiquinones leads to the generation of oxygen radicals, the corresponding laccase-catalysed oxidations yield water (Soden et al 2001). Fernandez-Larrea et al (1996) reported that the oxidative stress, which was caused by the presence of aromatic compounds, in Podospora anserina typically was accompanied by the induction of laccase mRNA. It recently has been reported by Soden that xenobiotic response elements (XREs) are present in the region upstream from the promoter of lac4 in Pleurotus sajor-caju, which is regulated by aromatic compounds. The XRE has a consensus sequence TNGCGTG, which is the cis-acting element sufficient to stimulate transcription of genes in eukaryote by aromatic compounds (Rushmore et al 1991, Li et al 1992). It is interesting to note that the fact that the laccase II gene in Trametes pubescens is not induced by aromatic compounds may be due to the absence of putative XREs in the lap2 promoter of this gene. Therefore, it is interesting to clone the gene encoding laccase B from Trametes sp. AH28-2, especially the promoter region of the gene, to explore the mechanism of regulation of laccase synthesis by aromatic compounds. Although yields of laccase produced by microorganisms can be increased by selecting suitable aromatic compounds, it is unsafe to use high concentrations of such toxic aromatic compounds in practice. It will be of great significance if suitable microorganisms instead of aromatic inducers can be used to stimulate laccase synthesis in Trametes sp. AH28-2. Initial studies show that A. oryzae or G. trabeum, neither of which can synthesize laccase by themselves, cannot take the place of aromatic inducers to significantly stimulate Trametes sp. AH28-2 to synthesize laccase.
Now that, in the presence of 3,5-dihydroxytoluene, Trametes sp. AH28-2 can synthesize laccase isozyme laccase B in a more efficient way and because other miscellaneous proteins co-existing with laccase B are relatively fewer, laccase B can be purified to homogeneity by two-step chromatography. Molecular mass of purified laccase B is determined to be 74 kDa by SDS-PAGE, which is larger than that of laccase A from Trametes sp. AH28-2 (Xiao et al 2003). Molecular mass of purified laccase B contains about 25% carbohydrates; thus, the larger molecular mass can be attributed to higher glycosylation. The kinetic constants of laccase B are remarkably different from those of laccase A (Table IV). In addition, laccase B has a lower pI value, a lower optimal reaction temperature and a higher pH optimum, when guaiacol is used as substrate. In addition, the thermal stability of laccase B apparently is inferior to that of laccase A. Laccase B will lose 50% of its activity if kept at 60 C for 14 min, whereas laccase A can retain almost all of its activity. It also can be proved that laccase B is different from laccase A by analyzing N-terminal amino acid sequence of the proteins: The N-terminal amino acid sequence of laccase A is AIGPTADLTISNAEV, which is the same as that of phenoloxidase from Coriolus hirsutus, and shares 87% similarity to the corresponding segment in the phenoloxidase gene (Xiao et al 2003), but the N-terminal amino acid sequence of laccase B is identical to the amino terminus of the lcc5 mature protein from T. villosa (Yaver et al 1996a). Therefore, laccase B is not a modified form of the gene encoding laccase A. At present, five laccase isozyme genes from T. villosa have been cloned but there is no detailed report on lcc5 mature protein. So the property comparison and similarity in the gene level between laccase B and lcc5 mature protein remains to be studied.
In conclusion, the results presented here indicate that, for Trametes sp. AH28-2 cultured in liquid medium, the total laccase activity is affected not only by the different concentrations of inducers added but also by the different type of inducers. Above all, the composition of laccase isozyme also is affected by the structures of inducers. It becomes more convenient to produce and purify laccase isozyme laccase B because it can be directly synthesized by induction of the inducer of certain structure in liquid medium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 31, 2003.
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LITERATURE CITED |
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- cocultured with A. oryzae in the presence of 4 mM o-toluidine. -
- cocultured with G. trabeum in the presence of 4 mM o-toluidine. -
- cocultured with A. oryzae without presence of o-toluidine. -+- cocultured with G. trabeum without presence of o-toluidine